Signal transduction pathways that act through heterotrimeric G proteins mediate the response to a wide variety of extracellular signals in many different organisms. In yeast, the pheromone response pathway is activated by the binding of extracellular pheromones to G protein- coupled receptors that are specific to cells of either the MATa or MATalpha mating type. Activation of the G protein alpha-subunit results in release of the beta-gamma-subunits, which transmit the signal to downstream kinases. Studies described in this proposal will investigate a novel function of Ste3p, the a-factor receptor. This novel function, called receptor inhibition, causes a block in signal transmission by inhibiting the activity of the beta-subunit in a manner that is independent of the alpha-subunit. Receptor inhibition only occurs when cells contain both a- specific and alpha-specific proteins, and probably functions immediately after the fusion of two mating cells to ensure that the signaling pathway is rapidly turned off. This process requires the product of a newly identified a-specific gene called ASG7. Thus, when the same cell expresses both Asg7p, an a-specific protein, and Ste3p, an alpha-specific protein, a process is initiated that acts directly on the beta-subunit and inhibits its activity. This project will investigate how beta-subunit signaling is inhibited when the a-factor receptor and the novel regulator Asg7p are present in the same cell. One specific aim is to determine how the activity of the beta- subunit is blocked by this process. The effect of changes in Asg7p abundance, localization, and binding to the beta-subunit will be tested for their effects on beta-subunit activity. A screen for other genes required for receptor inhibition will also be carried out. A second specific aim is to determine how Asg7p is activated by the presence of the Ste3p receptor. The potential roles of membrane localization, posttranslational modification, and Ste3p binding in the activation of As97p will be determined. A final specific aim is to investigate the structure/function relationships of Ste3p with respect to its receptor inhibition function. By isolating specific mutations in STE3 and by constructing chimeric receptors, the region of Ste3p involved in receptor inhibition will be identified. Significant progress in elucidating signal transduction mechanisms has been made in organisms in which sophisticated genetic manipulations can easily be performed. Elucidation of the process of receptor inhibition in yeast is likely to provide information about the regulation of G protein beta-subunits in other systems in which the beta-subunit plays an active role in signal transmission.